Our renewal application is a continuing evolution of studies aimed at identifying mechanisms that regulate TLR function and inflammation during sepsis. The overall goal of this proposal is to define CXCL12/chemokine receptor activation of G?i2 protein and ?-arrestin 1 and 2 signaling pathways that regulate crucial host defense responses to polymicrobial sepsis. During the last funding cycle a series of novel findings have given rise to the new focus. Our studies have established the importance of G?i2 protein as a negative regulator of TLR and sepsis-induced inflammation. We have also identified ?-arrestin proteins as negative regulators of TLR and sepsis-induced inflammation. Both G?i2 (-/-) mice and ?-arrestin 2(-/-) mice are highly susceptible to polymicrobial sepsis. However endogenous ligands and receptors that activate G?i2 and ?-arrestin signaling pathways that are beneficial in sepsis were uncertain. New findings have directed a focus on the CXCL12/chemokine receptor system as crucial endogenous regulators. CXCL12 binds to CXCR4 and activates canonical G?i2 signaling pathways. CXCL12 also binds CXCR7 which signals uniquely through ?-arrestin -dependent signaling pathways. We have recently demonstrated that severe sepsis suppresses endogenous CXCL12 production. Administration of a CXCL12 stable peptide surrogate, CTCE-0214 increases survival in severe CLP-induced polymicrobial sepsis and recruits peripheral mobilization of neutrophils and endothelial progenitor cells (EPCs). The enhanced mobilization of neutrophils correlated with greatly accelerated bacterial clearance in peritoneum, blood and lung and survival. We have also demonstrated that a CXCL12 mimetic is a potent suppressor of TLR and sepsis-induced inflammation. These novel findings provide the foundation of our overriding hypothesis that activation of the CXCL12/Chemokine receptor axis transduces G?i2 protein and ?-arrestin isoform-dependent signaling pathways critical in host defense responses to polymicrobial sepsis. Two interrelated specific aims will test this hypothesis. Specific Aim 1 will determine the receptor specificity, G?i protein and ?-arrestin isoform signaling dependence, and efficacy of a stable CXCL12 peptide surrogate in polymicrobial sepsis. Specific Aim 2 will determine agonist activated chemokine receptor and TLR4 signaling protein interactions, and G?i2 protein and ?-arrestin isoform dependence of these signaling pathways regulating neutrophil function. New knowledge derived from these studies may reveal innovative therapeutic strategies to treat polymicrobial sepsis that will have high translational value.